1. Field of the Invention
The invention relates in general to an improved method and apparatus for carrying out colorimetric, fluorimetric or photometric enzymatic assays which employ absorbent materials. In particular, this invention relates to the use of a superabsorbent polymeric material as the absorbent detecting layer in enzymatic assays which employ an enzyme reaction to produce a change in the spectral properties of a particular substance which is detectable by means of a suitable detecting device in order to enhance the sensitivity and detecting capacity of the assay.
2. Description of the Prior Art
In the detection of small molecules such as drugs, environmental toxins, and other compounds, it is quite common to use slide technology which primarily employs competitive binding assays using an antibody and an enzyme-labelled drug. Depending upon the design of the assay, several wash steps are usually employed, after which the enzyme substrate is added. Most of these assays operate by the formation of a color after a period of time whose intensity varies depending on the concentration of the detected drug in the test fluid. Still other assays operate by assessing fluorescence or reactions producing chemical luminescence. With most of these assays, the color change or photoluminescence will develop in an absorbent detecting layer which is generally a paper medium such as blotter paper. Unfortunately, however, blotter paper and similar materials are disadvantageous because they are opaque and have a low water absorbing capacity. Since the detection layer is opaque and the color or light formed is distributed throughout the medium, not all of the signal can be observed. Further, a low absorption capacity also means that less test fluid can be used, and thus less of the analyte is available for analysis. As a result, commonly used absorbent paper materials reduce the sensitivity of these enzymatic assays.
Alternative slide technologies are available, but these have disadvantages as well. In the Ektachem dry reagent system (see Curme et al., Clin. Chem. 24(8): 1335-1342 (1978)), agarose is used instead of blotter paper in the detection layer. In this system, the slide consists of three layers: a spreading layer, a polymer layer, and a protective layer. The test fluid such as a serum sample is applied through the spreading layer and diffuses into the polymer layer wherein an enzymatic reaction produces a colored dye whose concentration is proportional to the glucose concentration in the test fluid. This system is disadvantageous because of the slow hydration of the agarose polymer which therefore requires a complex spreading layer for uniform fluid distribution.
Superabsorbent polymers are a class of materials that are unique with regard to their water absorbing abilities. These polymers can absorb up to at least about 2,000 times their weight in water. The use of superabsorbent polymers as the absorbent material in the detecting layer of the colorimetric or photometric assays referred to above would be highly advantageous because these polymers can become hydrated much more quickly, can form colorless and transparent gels, and require little or no control of the amount of fluid used because excess fluid (beyond that which is absorbed) will diffuse out of the reaction area and no color will be produced.
It is thus highly desirable to develop a system by which superabsorbent polymeric materials can be used to improve the sensitivity of competitive binding assays in which an absorbent detecting layer is used in a colorimetric or photometric enzymatic analysis.